monosynaptic retrograde tracing of neurons expressing … · monosynaptic retrograde tracing of...

R E S E A R CH AR T I C L E

Monosynaptic retrograde tracing of neurons expressing theG-protein coupled receptor Gpr151 in the mouse brain

Jonas Broms1 | Matilda Grahm1 | Lea Haugegaard1 | Thomas Blom2 |

Konstantinos Meletis3 | Anders Tingstr€om1

1Psychiatric Neuromodulation Unit,

Department of Clinical Sciences, Faculty of

Medicine, Lund University, Lund, Sweden

2Biomedical Services Division, Faculty of

Medicine, Lund University, Lund, Sweden

3Department of Neuroscience, Karolinska

Institute, Stockholm, Sweden

Correspondence

Anders Tingstr€om, Psychiatric

Neuromodulation Unit, Biomedical Center,

D11, Klinikgatan 30, Lund 221 84, Sweden.

Email: [email protected]

Funding information

Swedish Research Council; Royal

Physiographic Society of Lund;

Vetenskapsrådet; Stiftelsen Professor Bror

Gadelius minnesfond; Kungliga

Fysiografiska Sällskapet i Lund;

Gyllenstiernska Krapperupsstiftelsen;

Stiftelsen Ellen och Henrik Sj€obrings

minnesfond

AbstractGPR151 is a G-protein coupled receptor for which the endogenous ligand remains unknown. In

the nervous system of vertebrates, its expression is enriched in specific diencephalic structures,

where the highest levels are observed in the habenular area. The habenula has been implicated in

a range of different functions including behavioral flexibility, decision making, inhibitory control,

and pain processing, which makes it a promising target for treating psychiatric and neurological

disease. This study aimed to further characterize neurons expressing the Gpr151 gene, by tracing

the afferent connectivity of this diencephalic cell population. Using pseudotyped rabies virus in a

transgenic Gpr151-Cre mouse line, monosynaptic afferents of habenular and thalamic Gpr151-

expressing neuronal populations could be visualized. The habenular and thalamic Gpr151 systems

displayed both shared and distinct connectivity patterns. The habenular neurons primarily received

input from basal forebrain structures, the bed nucleus of stria terminalis, the lateral preoptic area,

the entopeduncular nucleus, and the lateral hypothalamic area. The Gpr151-expressing neurons in

the paraventricular nucleus of the thalamus was primarily contacted by medial hypothalamic areas

as well as the zona incerta and projected to specific forebrain areas such as the prelimbic cortex

and the accumbens nucleus. Gpr151 mRNA was also detected at low levels in the lateral posterior

thalamic nucleus which received input from areas associated with visual processing, including the

superior colliculus, zona incerta, and the visual and retrosplenial cortices. Knowledge about the

connectivity of Gpr151-expressing neurons will facilitate the interpretation of future functional

studies of this receptor.

K E YWORD S

habenula, rabies, thalamus, RRID: IMSR_JAX:000664, RRID: IMSR_JAX:024109, RRID:

AB_10743815, RRID: AB_2571870, RRID: SCR_003070

1 | INTRODUCTION

The habenula is a paired epithalamic brain region whose structure and

connectivity are largely conserved throughout the vertebrate subphy-

lum (Bianco & Wilson, 2009; Díaz, Bravo, Rojas, & Concha, 2011;

Stephenson-Jones, Floros, Robertson, & Grillner, 2011). In mammals, it

can be further divided into two major subnuclei designated the medial

and lateral habenula, homologous to the dorsal and ventral habenula in

fish (Amo et al., 2010). In the mouse brain, the medial habenula receives

input from the medial septal nucleus, the triangular nucleus of septum,

the nucleus of the diagonal band, the bed nucleus of the anterior com-

missure, and the septofimbrial nucleus (Qin & Luo, 2009). The efferent

axons from the medial habenula form the core of the fasciculus retro-

flexus fiber bundle and terminate in the interpeduncular nucleus. The

afferent projections to the lateral habenula have not been investigated

in detail in the mouse brain. In rats however, the most prominent.......................................................................................................................................................................................This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any

medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.VC 2017 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

J Comp Neurol. 2017;525:3227–3250. wileyonlinelibrary.com/journal/cne | 3227

Received: 7 November 2016 | Revised: 16 June 2017 | Accepted: 19 June 2017

DOI: 10.1002/cne.24273

The Journal ofComparative Neurology

http://orcid.org/0000-0003-2416-1768

http://creativecommons.org/licenses/by-nc-nd/4.0/

afferent connections arise in the lateral preoptic area, the entopeduncu-

lar nucleus and the lateral hypothalamic area, and to a minor extent in

the lateral septal nucleus, the nucleus of the diagonal band, the prefron-

tal cortex, the median raphe, and the ventral tegmental area (Greatrex

& Phillipson, 1982; Herkenham & Nauta, 1977; Vertes, Fortin, & Crane,

1999; Warden et al., 2012). Fibers from the lateral habenular neurons

travel in the outer layer of fasciculus retroflexus and terminate primarily

in the hypothalamus, the ventral tegmental area, the rostromedial teg-

mental nucleus, the rhabdoid nucleus, the median and dorsal raphe

nuclei, and the pontine central gray (Broms, Antolin-Fontes, Tingstr€om,

& Iba~nez-Tallon, 2015; Quina et al., 2014).

The habenula has been implicated in several apparently distinct

brain functions, such as maternal behavior (Corodimas, Rosenblatt, &

Morrell, 1992), aggressive behavior (Chou et al., 2016; Golden et al.,

2016), social play (van Kerkhof, Damsteegt, Trezza, Voorn, & Vander-

schuren, 2013), drug seeking (Jhou et al., 2013), noxious substance

aversion (Donovick, Burright, & Zuromski, 1970; Fowler & Kenny,

2013), decision making (Stopper & Floresco, 2013), behavioral flexibil-

ity (Baker, Raynor, Francis, & Mizumori, 2016), and pain modulation

(Shelton, Becerra, & Borsook, 2012). Subdivision of the habenular com-

plex into smaller regions, defined by the expression of certain mole-

cules or neuronal connectivity patterns, is a necessary strategy for

deepening our understanding of its role in physiology and disease. This

approach has been useful to target neuronal subpopulations and inves-

tigate their anatomical and functional features, especially in the medial

habenula (Chou et al., 2016; Gardon et al., 2014; Hsu, Morton, Guy,

Wang, & Turner, 2016; Hsu et al., 2014; Kobayashi et al., 2013).

G-protein coupled receptor 151 (GPR151; also known as PGR7,

GALR4, GPCR-2037, and galanin-receptor like) is an orphan receptor

phylogenetically related to the galanin receptor family (Berthold, Collin,

Sejlitz, Meister, & Lind, 2003; Ignatov, Hermans-Borgmeyer, & Schaller,

2004; Vassilatis et al., 2003). In this work, we use capital letters to refer

to GPR151 protein while the term Gpr151 is used for mRNA, promoter

or gene. In a previous study using immunohistochemistry, we found

that the protein expression of GPR151 was limited to habenular neu-

rons targeting the interpeduncular nucleus, the rostromedial tegmental

nucleus, the rhabdoid nucleus, the median raphe, the caudal dorsal

raphe, and the dorsal tegmentum in rats and mice (Broms et al., 2015).

The protein expression pattern was similar in zebrafish, with strong

expression in habenular projections to the interpeduncular nucleus and

ventral median raphe, and this distinct and phylogenetically conserved

expression pattern suggests an important role of GPR151 in modulat-

ing habenular function.

Weak Gpr151 mRNA expression can also be detected in various

thalamic areas such as the paraventricular, the reunions, the rhomboid,

the central lateral, and the parafascicular nuclei (Ignatov et al., 2004;

Lein et al., 2006; Wagner, French, & Veh, 2014). Surprisingly, these

structures display no or very weak immunohistochemical staining of

GPR151 protein (Broms et al., 2015). In a recent study, specific ablation

of adult neurons exhibiting Gpr151 promoter activity resulted in several

behavioral alterations, such as increased anxiety, inability to habituate

to repeated exposure to a novel environment, increased impulsivity,

impaired pre-pulse inhibition, and spatial memory deficits (Kobayashi

et al., 2013). Decision making was also affected, where lesioned mice

preferred effortless and immediate small rewards over larger rewards

that required the animals to either wait longer for the reward or to

climb an obstacle. The observed behavioral changes were accompanied

by structural and neurochemical remodeling of the habenulo-

interpeduncular system. The authors concluded that the Gpr151-

expressing neurons may play an important role in inhibitory control and

cognition-dependent executive functioning (Kobayashi et al., 2013).

In addition to the expression in the above mentioned juxtaposed

diencephalic brain regions of the brain, Gpr151 mRNA is also detected

in certain sensory ganglia and cranial nerve nuclei (Ignatov et al., 2004).

In the spinal nerve ligation model of chronic neuropathic pain and in a

model of heat induced pain, the expression of Gpr151 mRNA was

strongly upregulated in dorsal root ganglia (Reinhold et al., 2015; Yin,

Deuis, Lewis, & Vetter, 2016). Gpr151 mRNA expression is also

increased in the facial nucleus of the brainstem in response to damage

of the facial nerve (Gey et al., 2016). These findings suggest that

GPR151, besides having a possible role in modulation of habenulo-

thalamic functions, might also be important for pain modulation in cra-

nial and peripheral nerves. However, a recent study utilizing mice carry-

ing a Gpr151 loss-of-function mutation could not confirm this

hypothesis (Holmes et al., 2017).

In a previous investigation (Broms et al., 2015), we characterized

the efferent connectivity of habenular neurons expressing the GPR151

protein using immunohistochemistry. The aim of the current study was

to identify the afferent regions projecting to Gpr151-expressing neu-

rons using monosynaptic pseudotyped rabies virus tracing, to gain fur-

ther knowledge about this diencephalic neuronal subpopulation. Using

pseudotyped rabies virus, Gpr151-expressing neurons can be targeted

specifically, avoiding some of the problems associated with conven-

tional tracers, such as uptake in fibers of passage and diffusion of tracer

to neighboring structures (Watabe-Uchida, Zhu, Ogawa, Vamanrao, &

Uchida, 2012; Wickersham et al., 2007). A detailed anatomical charac-

terization of the input and output of Gpr151-expressing neurons could

facilitate future functional studies of this orphan receptor.

2 | MATERIALS AND METHODS

2.1 | Nomenclature

In this article, we will primarily use the nomenclature and area delinea-

tions (Table 1) of Franklin and Paxinos (2008). We use the nomencla-

ture of Wagner, Stroh, and Veh, (2014) for the subnuclear organization

of the habenula. The categorization of brain areas was done according

to the hierarchy of the Allen Mouse Brain Atlas (Lein et al., 2006).

2.2 | Animals

All procedures described in the current investigation been approved by

the Malm€o-Lund Ethical Committee for the use and care of laboratory

animals (permit no. M37-16). The Tg(Gpr151-Cre)#Ito transgenic

mouse line containing random insertion of bacterial artificial chromo-

some (BAC) clone MSMg01-81G4 (Kobayashi et al., 2013) was kindly

provided by Dr. Itohara (Laboratory for Behavioral Genetics, RIKEN

3228 | The Journal ofComparative Neurology

BROMS ET AL.

Brain Science Institute, Saitama, Japan) and maintained on a C57BL/6J

background (RRID:IMSR_JAX:000664). To identify individual mice car-

rying the Cre recombinase allele, polymerase chain reaction genotyping

was performed (Transnetyx, Cordova, TN) using the proprietary “CRE”

probe set.

A total number of 46 adult (11–14 weeks old) mice was used in

the experiment. Wild type litter mates (Cre-negative) were used as

negative controls. The mice were housed in groups of two to six ani-

mals with free access to standard lab chow and water, in an air condi-

tioned room at 22–238C with a standard 12-hr light/dark cycle (lights

on 07:00 a.m., off 07:00 p.m.). The cages (1284L Eurostandard Type II

L, Techniplast, Italy) were enriched with aspen wooden bedding and

nesting material.

2.3 | Retrograde Cre-dependent tracing usingpseudotyped rabies virus

Monosynaptic tracing of the afferents of Cre-expressing neurons (Call-

away & Luo, 2015; Watabe-Uchida et al., 2012) was carried out in two

steps. First, Cre-dependent expression of an avian sarcoma-leukosis

virus receptor (TVA) fused to the fluorescent protein mCherry (TVA-

mCherry) and rabies glycoprotein (RG) was achieved by an intracerebral

injection of adeno-associated virus (AAV) vectors. Second, SADDG-

eGFP(EnvA), a glycoprotein-deleted (DG) rabies vector pseudotyped

with avian sarcoma-leukosis virus envelope protein (EnvA) was

injected. In Cre-expressing neurons, TVA-mCherry then becomes

expressed and acts as an entry receptor for the EnvA coated rabies

vector, while infection should not be possible in TVA-negative neurons.

RG allows budding of the viral particle and transsynaptic transport to

afferent neurons. The rabies vector also carried the gene for enhanced

green fluorescent protein (eGFP), allowing detection of infected neu-

rons. We will hereafter refer to Cre-positive and TVA-mCherry/RG-

expressing neurons that were infected by SADDG-eGFP(EnvA) as

starter neurons. The eGFP-positive/TVA-mCherry-negative afferent

neuronal populations lack expression of RG which prohibits further

propagation of the rabies virus (Figure 1).

The three AAV vectors, AAV8-Ef1a-FLEX-TVA-mCherry, AAV8-

CA-FLEX-RG, and AAV8-hSyn1-FLEX-mCherry, were obtained from

the UNC Vector Core (University of North Carolina at Chapel Hill, NC).

The FLEX-switch makes these three vectors Cre-dependent by allow-

ing one stable Cre-mediated inversion of the reversed transgene

sequence (Atasoy, Aponte, Su, & Sternson, 2008). SADDG-eGFP(EnvA)

was obtained from the Gene Transfer, Targeting and Therapeutics

Core (GT3) at Salk Institute (La Jolla, CA) and from Dr. Meletis at Karo-

linska Institute (Stockholm, Sweden).

Gpr151-Cre mice (n538) and wild type controls (n55) were

anesthetized with isoflurane (induction at 5% and maintenance at 1–

2%) and placed in a stereotaxic frame. The scalp was locally anesthe-

tized with bupivacaine (0.25%) and the skull exposed. After adjusting

the skull to a horizontal position, a hole was drilled through the skull,

exposing the brain surface. Using a thin glass pipette, 0.6 ml of a 1:1

mixture of AAV8-Ef1a-FLEX-TVA-mCherry (5.4 3 1012 viral genomes/

ml) and AAV8-CA-FLEX-RG (2.4 3 1012 viral genomes/ml) was

injected unilaterally into the habenula at two different anterior-

posterior levels (coordinates anteroposterior: 21.75, mediolateral:

10.35 or 20.35, dorsoventral: 22.7 and anteroposterior: 21.4, medio-

lateral: 10.35 or 20.35, dorsoventral: 22.7 relative to Bregma), the

paraventricular thalamic nucleus (anteroposterior: 20.46, mediolateral:

10.1 or 20.1, dorsoventral: 23.25 relative to Bregma), or the lateral

posterior thalamic nucleus (anteroposterior: 21.7, mediolateral: 11.3

or 21.3, dorsoventral: 22.7 relative to Bregma). In three of the animals

injected in the habenula, AAV8-CA-FLEX-RG was omitted to verify

that eGFP expression in neurons outside the injected area is a result of

transsynaptic transport of rabies virus particles rather than direct infec-

tion of afferent terminals. After the injection, the pipette was left in

the brain for 4 min to minimize back flow of the vector into the needle

tract. The skin was sutured with absorbable suture, and the mouse was

kept on a heat pad until recovered from the anesthesia, before

returned to its home cage.

After 21 days, the stereotaxic procedure was repeated in order to

inject the animals with 0.5 ml of the SADDG-eGFP(EnvA) vector (1.65–

3.0 3 107 transforming units/ml). Seven days after the last vector

injection, the mice were deeply anesthetized with an overdose of

sodium pentobarbital and transcardially perfused with isotonic sodium

chloride solution (0.9%) followed by cold phosphate buffered saline

(PBS; 0.1M sodium phosphate buffer with 0.15M sodium chloride) con-

taining 4% paraformaldehyde for 8 min (�100 ml per animal).

2.4 | Immunohistochemistry

The brains were dissected and postfixed in 4% paraformaldehyde for

18 hr at 48C. After post-fixation, the brains were immersed in sucrose

FIGURE 1 Distribution of fluorescent reporters in the Cre-dependent rabies virus retrograde tracing system. Cre-expressingneurons infected by AAV8-Ef1a-FLEX-TVA-mCherry express thesarcoma-leukosis virus receptor (TVA) fused to the fluorescent pro-tein mCherry (TVA-mCherry; magenta). TVA enables entry ofEnvA-coated pseudotyped glycoprotein-deleted rabies virus par-ticles (SADDG-eGFP(EnvA)) into Cre-positive neurons. Coinfectionof these neurons with AAV8-CA-FLEX-RG leads to expression ofrabies glycoprotein (RG) which enables transsynaptic retrogradetransport of the rabies virus particles. Both the Cre-positive starterneurons and the primary afferent neurons will thus express theenhanced green fluorescent protein (eGFP; green) while TVA-mCherry expression will be limited to Cre-positive neurons

BROMS ET AL. The Journal ofComparative Neurology

| 3229

solution (25% in PBS) at 48C for 1–3 days until equilibration had

occurred (i.e., the brains sank to the bottom of the vial). The brains

were frozen on dry ice, sectioned in 30 mm sections using a sliding

microtome (Thermo Scientific HM450) and cryoprotected in antifreeze

solution (30% glycerol, 30% ethylene glycol, 40% 0.5M PBS) before

storage at 2208C. During sectioning, the left hemisphere was marked

with a needle to keep track of the orientation of the sections.

Immunohistochemistry was performed on free floating brain sec-

tions as described previously (Broms et al., 2015). The sections were

washed in PBS, blocked with normal donkey serum (5%) in PBST (PBS

containing 0.05% Tween-20) for 1 hr at room temperature and subse-

quently incubated in blocking solution with primary antibody over night

at 48C. The primary antibodies used in the experiment are described

below. After repeated washing in PBS, the sections were incubated

with biotinylated or Cy5-conjugated secondary antibodies at 3 mg/ml

(Jackson ImmunoResearch Europe Ltd., Suffolk, United Kingdom) in

PBST, 2 hr at room temperature. For immunofluorescent detection,

sections were washed and mounted on Superfrost Plus glass slides

(Menzel Gläser, Brunswick, Germany) with a polyvinyl alcohol and glyc-

erol based mounting solution containing the anti-fading agent 1,4-dia-

zabicyclo[2.2.2]octane (PVA-DABCO).

Fluorescent images were acquired using a Nikon Eclipse Ti confo-

cal microscope with NIS-Elements (version 4.40) software (Nikon

Instruments Europe BV, Amsterdam, The Netherlands). For automatic

detection and marking of eGFP-expressing neurons in scanned images,

ImageJ (version 2.0.0-rc-59/1.51n, RRID:SCR_003070) was used. The

eGFP channel was thresholded to create a binary mask. The positions

of eGFP-expressing cells were then obtained using the “Analyze Parti-

cles. . .” subroutine of ImageJ. Each location was marked with a green

circle. A grayscale look-up table was applied to the mCherry channel

and the background fluorescence of this channel provided a back-

ground for each micrograph.

For light microscopy detection, biotinylated secondary antibodies

were used. After washing in PBS, the sections were incubated with

avidin-biotin-peroxidase complex (diluted 1:125 in PBS) (VECTASTAIN

Elite ABC Kit, Vector Laboratories, Burlingame, CA) and developed using

3,3’-diaminobenzidine (DAB, 0.5 mg/ml) with nickel chloride (0.5 mg/

ml). Following the DAB reaction, sections were mounted, dehydrated in

increasing concentrations of ethanol followed by xylene and finally cov-

erslipped with p-xylene-bis-pyridinium bromide (DPX) (Fisher Scientific,

Pittsburgh, PA). Low resolution micrographs were acquired using a slide

scanner (PathScan Enabler 5, Meyer Instruments Inc., Houston, TX).

2.5 | Antibody characterization

Rabbit GPR151 polyclonal antibody (SAB4500418; Sigma-Aldrich, St.

Louis, MO; RRID: AB_10743815) was raised against a synthetic pep-

tide containing amino acids 370-419 (EKEKPSSPSSGKGKTEKAEI-

PILPDVEQFWHERDTVPSVQDNDPIPWEHEDQETGEGVK) of the C-

terminal of human GPR151. It recognizes a single band of 46 kDa on

western blots of lysate of GPR151-expressing K562 cells. The specific-

ity was confirmed by pre-adsorption with the immunogen peptide

(according to the manufacturer’s technical information). In homozygous

Gpr151-knockout mice, the immunoreactivity was completely abol-

ished, further proving the specificity of the antibody (Broms et al.,

2015). In the present experiment, a working concentration of 0.125

mg/ml was used.

Rabbit mCherry polyclonal antibody (ab167453; Abcam, Cam-

bridge, MA; RRID:AB_2571870) was raised against recombinant full

length mCherry protein (MVSKGEEDNMAIIKEFMRFKVHMEGSVN

GHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKA

YVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEF

IYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRL

KLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQ

YERAEGRHSTGGMDELYK), a fluorescent protein derived from the

DsRed protein. According to the manufacturer’s specifications, the

antibody recognizes a �27 kDa band in lysate of HEK293 cells trans-

fected with a pFin-Ef1a-mCherry vector. In our experiment, the anti-

body (used at a working concentration of 0.2 mg/ml) produced a

specific immunohistochemical signal in brains of Gpr151-Cre animals

injected with the AAV8-Ef1a-FLEX-TVA-mCherry vector.

A different set of Gpr151-Cre mice (n53) were injected bilaterally

in the habenula with AAV8-hSyn-FLEX-mCherry (0.6 ml, 6.1 3 1012

viral genomes/ml) to label cell bodies of Cre-expressing neurons. Four

weeks following the injections the brains were prepared for in situ

hybridization. Perfusion and postfixation in paraformaldehyde was car-

ried out as described above. After equilibration in 25% phosphate buf-

fered sucrose solution, 14 mm thick brain sections mounted onto

Superfrost Plus glass slides using a cryostat. Detection of Gpr151-

mRNA was performed using the RNAscope® Fluorescent Multiplex

Assay with a probe set targeting base pairs 27–988 of mouse Gpr151-

mRNA (Cat No. 317321, Advanced Cell Diagnostics, Inc., Newark, CA)

(Wang et al., 2012). Briefly, the sections were air dried for 20 min,

washed in PBS and subsequently immersed in boiling 1x Target

Retrieval solution for 5 min. After washing in distilled water and 100%

ethanol for 2 min each, the sections were incubated with Protease IV

solution for 30 min at 408C in a humidified tray. After washing in dis-

tilled water for 2 min, the sections were then hybridized with Gpr151

target probe for 2 hr at 408C followed by amplification solutions 1 (30

min), 2 (15 min), 3 (30 min), and 4 (15 min) at 408C. Between each

amplification step, the sections were rinsed in 1x Wash Buffer for 2

min at room temperature. Finally, the sections were counterstained

with 4’,6-Diamidine-2’-phenylindole dihydrochloride (DAPI) for 30 s

and coverslipped in PVA-DABCO. The fluorochrome used for detection

in this system was Alexa488. Images were acquired using a Nikon

Eclipse Ti confocal microscope with NIS-Elements software.

3 | RESULTS

3.1 | GPR151 protein and mRNA detected in

Cre-expressing neurons

As previously reported, immunofluorescent GPR151 staining was pro-

nounced in habenular axonal projections, while often barely above

background level in the neuronal somata (Broms et al., 2015). To better

visualize cell bodies with expression of Cre, we injected Gpr151-Cre


BROMS ET AL.

mice with AAV8-hSyn1-FLEX-mCherry in the habenular region

(Figure 2).

Colocalization between GPR151 protein and mCherry could be

observed in fibers in the fasciculus retroflexus (Figure 2d–i), which con-

tains the efferent axons from the habenula. In the habenula, mCherry-

expressing cell bodies were localized in the ventral division of the medial

habenula and in most lateral habenular subnuclei, excluding the oval

subnucleus of the lateral division of lateral habenula (Figure 2c). Further-

more, mCherry-positive neurons were also observed in the paraventric-

ular thalamic nucleus, and in the lateral dorsal and lateral posterior

thalamic nuclei. No GPR151 protein could be detected in these thalamic

areas using immunohistochemistry (Figure 2c; Broms et al., 2015).

Next, we performed fluorescent in situ hybridization using a RNA-

scope® probe targeting mouse Gpr151 mRNA on brain sections of

mice that had been injected with AAV8-hSyn1-FLEX-mCherry into the

habenula. In Figure 3, expression of Gpr151 mRNA in mCherry-

expressing neurons is seen in neurons located in the habenula, para-

ventricular thalamic nucleus and the lateral posterior thalamic nucleus.

The strongest Gpr151 mRNA expression was observed in medial and

lateral habenula, compared to much weaker expression in the paraven-

tricular thalamic nucleus while only a very faint expression was

observed in neurons of the lateral posterior thalamic nucleus. In the

habenula (Figure 3e) and the paraventricular thalamic nucleus, mCherry

colocalized with the Gpr151 mRNA signal. In the lateral posterior

FIGURE 2 GPR151 protein expression in axons of Gpr151-Cre neurons. Coronal sections of a Gpr151-Cre mouse injected with AAV8-hSyn1-FLEX-mCherry in the habenular region. Panels (g–i) represent magnifications of the insets in (d–f). Cell bodies positive for mCherry (magenta)were observed in the ventral medial habenula, the lateral habenula (excluding the oval subnucleus of the lateral division of lateral habenula), thelateroposterior, and paraventricular thalamic nuclei (b, c; 21.94 mm posterior to Bregma). Fibers expressing mCherry and GPR151 protein (green)exited the habenula through the fasciculus retroflexus (a–i; 22.30 mm posterior to Bregma), where colocalization between mCherry and GPR151was observed (arrows; g–i). Scale bar (a–f)5100 mm, (g–i)520 mm


| 3231

thalamic nucleus, Gpr151 was undetectable in some mCherry-positive

neurons (Figure 3d). This finding is further considered in the discussion.

Using a crossing between Gpr151-Cre mice and two different ß-

galactosidase reporter strains, Kobayashi et al. (2013) identified Cre-

expressing cells in the medial and lateral habenula, paraventricular tha-

lamic nucleus and the reuniens thalamic nucleus. This expression pat-

tern largely agrees with our findings, except that more Cre-expressing

cells were detected in the laterodorsal and lateral posterior thalamic

nucleus in the current investigation. Given that Gpr151 mRNA expres-

sion is accentuated in the habenula and paraventricular thalamic

nucleus (Ignatov et al., 2004; Lein et al., 2006), no viral injections

targeting the reuniens thalamic nucleus were performed in the current

investigation.

3.2 | Monosynaptic tracing of afferents to

Cre-expressing neurons

Using the Cre-dependent rabies virus tracing system, Gpr151-Cre

starter neurons were labeled with TVA-mCherry and eGFP, while

monosynaptic afferents to these starter neurons were labeled with

eGFP only. Not all TVA-mCherry-expressing neurons coexpressed

eGFP. This may be explained by different transduction efficiency of the

FIGURE 3 Gpr151 mRNA expression in Gpr151-Cre neurons. Coronal section of a Gpr151-Cre mouse injected with AAV8-hSyn1-FLEX-mCherry in the habenular region (21.82 mm posterior to Bregma). Gpr151 mRNA expression (green) can be seen in the ventral medialhabenula, lateral habenula (excluding the oval subnucleus of the lateral division), as well as in the lateral posterior, central lateral, and para-ventricular thalamic nuclei (panels a, c, d, e). Expression of mCherry (magenta, panels b–e) show partial overlap with Gpr151 mRNA. Trans-duction efficiency and injection localization could explain the lack of mCherry expression in certain Gpr151-positive subregions like the

LHbMS. Examples of coexpression of mCherry and Gpr151 mRNA in the lateral posterior thalamic nucleus and lateral habenula are shownin panel (d) and (e) (magnifications of insets in panel c). Scale bar (a–c)5100 mm, (d, e)520 mm


BROMS ET AL.

TABLE 1 Abbreviations

Abbreviation Area Abbreviation Area

3V 3rd ventricle Me medial amygdaloid nucleus

aca anterior commissure, anterior part MG medial geniculate nucleus

Acb accumbens nucleus MHb medial habenular nucleus

AD anterodorsal thalamic nucleus MHbD dorsal subnucleus of the MHb

AH anterior hypothalamic area MHbS superior subnucleus of the MHb

AI agranular insular cortex MHbV ventral part of the MHb

Arc arcuate hypothalamic nucleus MHbVc central subnucleus of the MHbV

Au auditory cortex MHbVl lateral subnucleus of the MHbV

AV anteroventral thalamic nucleus MHbVm medial subnucleus of the MHbV

BAC bed nucleus of the anterior commissure MnPO median preoptic nucleus

BL basolateral amygdaloid nucleus MnR median raphe nucleus

BM basomedial amygdaloid nucleus MPA medial preoptic area

CG cingulate cortex MPT medial pretectal nucleus

Cg1 cingulate cortex, area 1 MPtA medial parietal association cortex

Cg2 cingulate cortex, area 2 mRt mesencephalic reticular formation

CL centrolateral thalamic nucleus MS medial septal nucleus

CI claustrum MTu medial tuberal nucleus

CPu caudate putamen (striatum) Pa paraventricular hypothalamic nucleus

DB nucleus of the diagonal band PAG periaqueductal gray

DG dentate gyrus Pe periventricular hypothalamic nucleus

DLG dorsal lateral geniculate nucleus PF parafascicular thalamic nucleus

DM dorsomedial hypothalamic nucleus PG pregeniculate nucleus

DR dorsal raphe nucleus PH posterior hypothalamic nucleus

EP entopeduncular nucleus PLH peduncular part of lateral hypothalamus

f fornix PnO pontine reticular nucleus, oral part

fmi forceps minor of the corpus callosum Po posterior thalamic nuclear group

fr fasciculus retroflexus PrC precommissural nucleus

IC inferior colliculus PrL prelimbic cortex

IF interfascicular nucleus PT paratenial thalamic nucleus

IL infralimbic cortex PV paraventricular thalamic nucleus

PVA paraventricular thalamic nucleus, anterior part

IP interpeduncular nucleus PVP paraventricular thalamic nucleus, posterior part

LD laterodorsal thalamic nucleus RCh retrochiasmatic area

LDTg laterodorsal tegmental nucleus RLi rostral linear nucleus (midbrain)

LHb lateral habenular nucleus RMg raphe magnus nucleus

LHbA anterior division of the LHb RSD retrosplenial dysgranular cortex

LHbL lateral division of the LHb Rt reticular nucleus (prethalamus)

LHbLB basal subnucleus of the LHbL S1 primary somatosensory cortex

LHbLMc magnocellular subnucleus of the LHbL S2 secondary somatosensory cortex

(Continues)


| 3233

AAV and rabies vectors or that the vectors were injected at slightly dif-

ferent locations due to the margin of error of the stereotaxic technique.

The injections targeted one of three areas, the habenula, the paraventric-

ular thalamic nucleus, or the lateral posterior thalamic nucleus. The

starter neurons at the injection sites and the resulting eGFP expression

in afferent structures are summarized in Tables 2 and 3.

In wild type animals, occasional neurons expressing mCherry and

eGFP could be observed close to the injection tract in the habenula,

which implies minor local Cre-independent expression (Figure 4j–l;

Table 2). However, no eGFP1 neurons were detected outside of the

injected area, so the analysis of eGFP1 neurons far from the injected

site is not impacted.

SADDG-(EnvA) is capable of infecting neurons with minute expres-

sion of TVA (Callaway & Luo, 2015). Since it has been shown that trace

amounts of TVA-expression can result from retrograde transport of AAV-

TVA (Menegas et al., 2015), it is important to confirm that the starter neu-

rons are indeed restricted to the injected area. By omitting AAV8-CA-

FLEX-RG, transsynaptic transport is inhibited and eGFP expression should

be restricted to local TVA-expressing neurons. In Gpr151-Cre animals

where AAV8-CA-FLEX-RG was omitted, a strong expression of TVA-

mCherry and eGFP was only observed in the vicinity of the injection, but

not anywhere else in the brain (Figures 4g–i and 5; Table 2). This confirms

that any eGFP1 neurons found outside of the injected area result from

RG-dependent transsynaptic transport of rabies virus.

3.3 | Afferents to Gpr151-Cre neurons in the

habenula

Seven cases with good quality injections targeting the habenula were

considered for further analysis (Figure 4a–f; Table 2). In all cases, a

majority of starter neurons were detected in the habenula, although a

varying degree of spread to neighboring thalamic structures, such as

the paraventricular thalamic nucleus and the lateral posterior thalamic

nucleus was also observed.

Very few eGFP1 neurons were detected in the cerebral cortex

(Figure 6; Table 3). Occasional eGFP1 neurons were observed in

medial prefrontal areas, most notably bilaterally in the prelimbic and

infralimbic cortices.

Several structures in the pallidum contained large numbers of

eGFP1 neurons (Table 3). Moderate numbers of eGFP1 neurons were

detected in the medial (Figure 7a) and triangular septum (Figure 7d).

The nucleus of the diagonal band contained many eGFP1 neurons,

both in the vertical and horizontal limb (Figure 7a). Moderate numbers

of eGFP1 neurons were also observed in the bed nucleus of the ante-

rior commissure (Figure 7d). The bed nucleus of the stria terminalis

contained great numbers of eGFP1 neurons, especially in case M17

(Figure 7c). In two cases (M18 and M104), a dense network of eGFP1

neurons was seen in the entopeduncular nucleus (Figure 7e,f). Interest-

ingly, these specific cases were distinguished by a large number of

starter neurons in lateral division of the lateral habenula as well as

spread into the lateral posterior thalamic nucleus.

The hypothalamus was another major source of input to the

Gpr151-Cre neurons of the habenula (Table 3). A continuous group of

eGFP-expressing neurons stretching from the lateral preoptic area to

the peduncular part of the lateral hypothalamus was observed in all

cases (Figures 6 and 7b,e,f). Scattered neurons were also observed in

several medial hypothalamic nuclei (Table 3). In two cases (M18 and

M104), the same animals which displayed eGFP1 neurons in the ento-

peduncular nucleus, eGFP1 neurons could also be observed in the

zona incerta.

Because undetectable expression of TVA-mCherry may be suffi-

cient to allow entry of the rabies vector (Callaway & Luo, 2015), it

TABLE 1 (Continued)

Abbreviation Area Abbreviation Area

LHbLMg marginal subnucleus of the LHbL SC superior colliculus

LHbLO oval subnucleus of the LHbL SCh suprachiasmatic nucleus

LHbLPc parvocellular subnucleus of the LHbL SFi septofimbrial nucleus

LHbM medial division of the LHb SHy septohypothalamic nucleus

LHbMC central subnucleus of the LHbM sm stria medullaris

LHbMMg marginal subnucleus of the LHbM ST bed nucleus of the stria terminalis

LHbMPc parvocellular subnucleus of the LHbM TS triangular septal nucleus

LHbMS superior subnucleus of the LHbM V1 primary visual cortex

LHbP posterior division of the LHb V2 secondary visual cortex

LP lateral posterior thalamic nucleus VMH ventromedial hypothalamic nucleus

LPO lateral preoptic area VP ventral pallidum

LS lateral septal nucleus VTA ventral tegmental area

M medial mammillary nucleus ZI zona incerta

ME median eminence ZIR zona incerta, rostral part

Abbreviations of mouse brain regions according to Franklin and Paxinos (2008) and Wagner, Stroh, et al. (2014).


BROMS ET AL.

TABLE 2 Starter neuron populations

Habenula Paraventricular thalamic nucleus

M17 M18 M80 M102 M103 M104 M105 M74 M112 M113 M120 M121

Medial habenula MHbD

MHbS

MHbVl 11111 11111 111 11 11 1111

MHbVc 11111 11111 111 11 11 111

MHbVm 1 1

Lateral habenula LHbA 1 111 1 11 11 1

LHbMS 11111 11 11111 1111 11 1111

LHbMPc 1 111 11

LHbMC 11 11 111 11 111

LHbMMg 111 1

LHbLMg 11 11 11 1

LHbLPc 11 11 111 111 1111

LHbLMc 1 11111 111 11111 11111 11 11

LHbLB 1111 1111 1111 11

LHbLO

LHbP 1 1111 1111 111 11 1

Paraventricularthalamic nucleus

PVA 111 11111 11111 111 11111

PV 1 1 111 1 1 1 11 11 11111

PVP 1

Lateral thalamicnuclei

LP 11 11

LD 1 11

CL 1 1 1

Posteriorthalamic nuclei

PF

PrC 1

Po

MPT 1

Lateral posterior thalamic nucleus Habenula no RG Habenula wild type

M114 M115 M116 M119 M106 M117 M118 M113 M16 M77 M77 M78 M81

Medial habenula MHbD

MHbS

MHbV

MHbVc

MHbVm

Lateral habenula LHbA 11 11 11 1

LHbMS 111 111 111 1111 11 1

LHbMPc 1 11 1 1 1 11

LHbMC 111 11 11 111 11

(Continues)


| 3235

cannot be ruled out that eGFP1 neurons close to the injected area

that appear to lack mCherry are indeed afferent neurons and not

starter neurons. Because of the proximity of the injection target

area to some Gpr151-expressing thalamic nuclei, it makes interpreta-

tion of eGFP1 neurons in these areas uncertain. Scattered eGFP1

neurons were detected in the pregeniculate, dorsolateral geniculate,

and medial geniculate nuclei which are far from the injected area.

The parafascicular thalamic nucleus and the precommissural nucleus

also contained a small number of eGFP1 neurons, along with occa-

sional starter neurons. Occasionally, eGFP1 neurons were visible in

the medial or lateral habenula contralateral to the injected side

which suggests minor contribution from an intrahabenular

projection.

In the midbrain, scattered eGFP1 neurons were observed in the

pretectal area, superior colliculus, and the periaqueductal gray. A few

eGFP1 cells could also be detected in the interpeduncular nucleus,

which is the projection target of neurons in the medial habenula. This

implies a reciprocal connection between the habenular complex and

the interpeduncular nucleus. EGFP1 neurons were also observed in

many monoaminergic midbrain areas such as the ventral tegmental

area (primarily the interfascicular nucleus), rostral linear nucleus,

median, and dorsal raphe nuclei.

Very few eGFP1 neurons were detected in hindbrain areas,

including the laterodorsal tegmental nucleus, pontine reticular nucleus,

and raphe magnus nucleus.

3.4 | Afferents to Gpr151-Cre neurons in the

paraventricular thalamic nucleus

Five animals where starter neurons could be observed in the paraven-

tricular thalamic nucleus (Table 2) were further analyzed regarding

afferents (Figures 8 and 9; Table 3). The starter neuron population was

entirely restricted to the target area except for one case (M113) where

a few cells were also observed in the anterior division of the lateral

habenula.

Cortical afferents were restricted to frontal cortical areas such as

the prelimbic, infralimbic, and agranular insular cortices. In two cases

(M113 and M121), a few eGFP1 neurons could also be observed in

the claustrum.

The distribution of eGFP1 neurons in the pallidum was remarkably

similar to that of the afferents to the habenular Gpr151-Cre population

(Table 3). The nucleus of the diagonal band, bed nucleus of stria termi-

nalis, and the ventral pallidum contained many eGFP1 neurons. No

eGFP1 neurons were observed in the medial septal nucleus or the

TABLE 2 (Continued)

Lateral posterior thalamic nucleus Habenula no RG Habenula wild type

M114 M115 M116 M119 M106 M117 M118 M113 M16 M77 M77 M78 M81

LHbMMg 11

LHbLMg 11 111

LHbLPc 11 11 111 1111 111

LHbLMc 111 11 11111 1

LHbLB 1111

LHbLO

LHbP 111 11 111 11111 11111 11111

Paraventricularthalamic nucleus

PVA

PV 1 11

PVP 11 111

Lateralthalamic nuclei

LP 11111 11111 11111 11111 111 11

LD 11111 11111 11111 11111 1111

CL 1 11 111 1 111

Posteriorthalamic nuclei

PF 1 11 1111

PrC 1 1 11 111 111

Po 1111 11 1111

MPT 1 11 11 1 11

Semiquantitative estimate of neurons coexpressing TVA-mCherry and eGFP (starter neurons) in target areas and neighboring structures. Each area wasgraded from1 to 11111 depending on the number of mCherry/eGFP coexpressing cells observed.


BROMS ET AL.

TABLE3

Locations

ofprim

aryafferentsto

Gpr151-C

rene

urons.

Hab

enula

Parav

entricular

thalam

icnu

cleu

sLa

teralposteriorthalam

icnucleu

s

M17

M18

M80

M102

M103

M104

M105

M74

M112

M113

M120

M121

M114

M115

M116

M119

Cereb

ral

cortex

Al

1/1

Au

1/-

1/-

BL

1/1

BM

1/-

Cg1

1/-

1/-

1/-

11/-

111/-

111/-

11/-

Cg2

1/-

1/-

1/-

-/1

11/1

111/-

1/-

11/1

Cl

1/-

1/-

1/1

1/1

1/-

DG

1/-

1/-

1/-

IL1/-

1/1

1/-

1/-

-/1

1/-

1/1

11/-

MPtA

1/-

PrL

1/1

11/1

1/-

1/1

111/1

1-/1

11/1

1/-

1/-

11/1

111/1

RSD

1/-

1/-

111/-

S11/-

11/-

S21/-

V1

11/-

1/-

V2

11/-

1/-

1/-

Pallid

umBAC

111/

11

1/-

1/-

1/-

11/-

1/-

1/-

11/-

11/1

1

DB

111/

11

1111/

11111/

111/1

1/1

111/1

1111/1

11/1

111/-

1/1

11/

11

11/-

11/-

1/-

111/1

EP

11/-

111

11/1

11111/1

1/-

11/-

1/-

111/1

MS

11

111

11

11

11

ST1111/

111

111/-

11/-

1/-

111/-

11/1

111/1

11/-

1/1

1111/

11

111/

11111/

11

111/

11

TS

1/-

1/-

1/1

111/1

1/-

VP

11/1

111/-

11/1

1/-

11/-

111/1

111/1

1/1

1/-

-/1

1/1

111/1

11/-

1/-

1111/

11

Striatum

Acb

1/-

11/1

111/1

11/-

1/1

1/- (C

ontinues)


| 3237

TABLE3

(Continue

d)

Hab

enula

Parav

entricular

thalam

icnu

cleu

sLa


icnucleu

s

M17

M18

M80

M102

M103

M104

M105

M74

M112

M113

M120

M121

M114

M115

M116

M119

CPu

11/-

1/1

1/-

11/1

11/1

11/-

1/-

11/-

LS1/-

1/-

1/1

1/-

1/-

1/-

1/1

1/-

1/-

11/1

1/-

1/-

1/1

Me

1/-

1/-

Hyp

othalam

usAH

1/-

1/-

1/-

1/1

1/-

1/-

1/1

11/1

11/-

1/1

1/1

Arc

1/-

-/1

1/-

1/-

11/1

11/1

1/-

11/1

11/-

1/-

11/-

-/1

DM

11/-

1/-

1/-

1/1

11/1

111/

111

-/1

1/1

1/-

1/-

1/-

LPO

111/

11

1111/

11

1/-

11/1

11/1

11111/

11

11/-

1/1

1/1

11/1

11/1

1111/1

111/1

11111/1

1111/

11

M1/-

1/1

1/-

1/1

1/-

11/1

MnP

O1

11

11

11

MPA

-/1

1/1

11/-

1/-

11/1

11111/

111

1/1

11/

111

1/-

11/1

11/1

MTu

1/1

-/1

Pa

1/-

1/-

11/1

1/-

Pe

1/-

1/1

11/1

11/1

PH

-/1

1/-

1/-

1/-

PLH

111/

11

11111/

111

11/1

111/1

1111/

11

11111/

11

1111/

11

1/-

11/1

1111/1

11/-

11/

11

11111/

11

111/

11

11111/

111

11111/

111

RCh

1/1

1/1

11/-

111/1

1-/1

SCh

1/-

1/1

11/1

1/-

1/1

SFi

11/-

111/-

SHy

1/-

1/1

11/-

11/1

1

VMH

1/-

1/-

11/-

1/-

1/1

111/1

11

11/1

1/-

1/-

ZI

11/-

1/-

111/-

1/-

1/-

111/1

11

11111/

11111

11/1

111/

111

1111/-

11/-

1111/1

1111/1

Tha

lamus

AD

1/-

LD/LP

(contralateral

side

)

1

(Continues)


BROMS ET AL.

TABLE3

(Continue

d)

Hab

enula

Parav

entricular

thalam

icnu

cleu

sLa


icnucleu

s

M17

M18

M80

M102

M103

M104

M105

M74

M112

M113

M120

M121

M114

M115

M116

M119

LHb

(contralateral

side

)

11

11

MD

1/-

11/-

1/1

1/-

MG/P

G/D

LG1/1

1/-

1/-

11/1

1/-

11/-

1/-

111/1

111/1

MHb

(contralateral

side

)

11

1

PF

1/-

1/1

1/-

1/-

11/1

1/-

1/-

1/1

11/1

11/-

1/-

Po

111/-

PrC

1/-

1/-

1/-

11/-

1/-

1/-

PVA

1/-

11/-

1/-

1/1

1/-

111/1

11/-

1/-

Rt

11/-

-/1

1111/1

111/-

11111/-

11111/1

Midbrain

DR

1/-

1/-

1/-

11/1

11

1/-

1/1

11

1/1

11/1

11/-

IC1/-

IP1/1

1/-

-/1

1/1

11/-

1/-

11/1

MnR

11/1

1/-

1/-

1/1

1/1

11/1

11/1

1/-

1/1

11

11/1

MPT

11/-

11/-

11/1

111/

11

111/1

1

mRT

11/1

1/-

11/1

1/1

-/1

1/1

1/-

1/-

11/-

PAG

1/-

1/-

1/-

11/1

11/-

1/1

11/1

11/-

1/1

1/-

11/-

1/1

RLi

11

11

1

SC1/-

1/-

1/-

-/1

1/-

1/1

1/1

11111/1

1111/

11

11111/1

111111/

11

VTA

1/-

1/-

1/-

1/-

1/-

11/1

1/-

1/1

1/-

1/-

1/1

Hindb

rain

LDTg

1/-

1/-

1/-

1/-

11/-

PnO

1/1

1/-

1/1

1/-

1/-

RMg

1/-

1/-

Semiqua

ntitativeestimateofprim

aryafferentsto

Gpr151-C

rene

urons.The

entire

brainwas

inve

stigated

andarea

sweregrad

edfrom1

to11111

dep

endingonthenumber

ofeG

FPex

pressingcells

foun

d.In

paired

structures,theipsilateralestimateis

shownto

theleft

andtheco

ntralateralestimateto

therigh

t(ip

silateral/co

ntralateral).


| 3239

FIGURE 4 Expression of TVA-mCherry and eGFP after injections of viral vectors at target sites. Coronal sections through the target areasof cases M80 (habenula; a–c), M18 (habenula; d–f), M118 (habenula, no RG; g–i), M77 (habenula, wild type; j–l), M119 (lateral posterior tha-

lamic nucleus; m–o), and M113 (paraventricular thalamic nucleus; p–r) showing sarcoma-leukosis virus receptor (TVA) fused to the fluores-cent protein mCherry (TVA-mCherry; magenta; b, e, h, k, n, q), eGFP expression (green; a, d, g, j, m, p), and an overlay between the two (c,f, i, l, o, r). Scale bar5100 mm


BROMS ET AL.

entopeduncular nucleus however, in contrast to the afferents of the

Gpr151-Cre neurons in the habenula.

In the striatum, moderate numbers of eGFP1 neurons were detected

in the accumbens nucleus, caudate putamen, and the lateral septum.

The main source of input to the paraventricular thalamic Gpr151-

Cre population was the hypothalamus (Table 3). Similarly to the habe-

nular Gpr151-Cre population, eGFP1 neurons could be detected in

the lateral preoptic area and the peduncular part of the lateral hypo-

thalamus (Figure 9e). Great numbers of eGFP1 neurons were also

seen in the medial preoptic area, and to a lesser extent the median pre-

optic area (Figure 9b). Medial hypothalamic areas such as the anterior,

ventromedial, dorsomedial, arcuate, paraventricular, and periventricular

hypothalamic nuclei also contained many eGFP1 neurons (Figure 9b,d,

e,f). Some afferent neurons were also seen in the retrochiasmatic (Fig-

ure 9d) and suprachiasmatic nuclei. The strongest projection arose

from a cluster of cells in the rostral part of the zona incerta (Figure 9c).

Some eGFP1 neurons were also detected in thalamic areas such

as the parafascicular and mediodorsal thalamic nuclei. In the midbrain,

there were also a small number of eGFP1 neurons in the periaqueduc-

tal gray and the mesencephalic reticular formation.

3.5 | Efferent projections of the Cre-expressing

population in the paraventricular thalamic nucleus

Since the paraventricular thalamic Gpr151-Cre population could be tar-

geted specifically without spread into other Gpr151-expressing popula-

tions, we decided to also analyze the efferent connectivity of this

population. Immunohistochemistry using mCherry antiserum was per-

formed to visualize the efferent projections of the TVA-mCherry-

expressing neurons (Figure 10). The cell bodies of these neurons were

confined to the paraventricular nucleus, while dense fields of mCherry-

immunoreactivity could be observed in the prelimbic area, the shell and

core regions of accumbens nucleus, the basolateral amygdala, and the

zona incerta. It should be noted that while no GPR151 protein could

be detected by immunohistochemistry in any of these areas (Broms

et al., 2015), Gpr151 mRNA was observed in the paraventricular tha-

lamic nucleus in the current study (Figure 3c) and has also been

reported previously (Ignatov et al., 2004; Wagner, French, et al., 2014).

3.6 | Afferents to Gpr151-Cre neurons in the lateral

posterior thalamic nucleus

Four animals with injections targeting the lateral posterior thalamic

nucleus were analyzed (Figure 11; Table 3). Large quantities of starter

neurons were observed in a group of neurons in the lateral dorsal and

lateral posterior thalamic nuclei. In two cases, the labeled population

also extended into the posterior thalamic group (M114 and M119).

Unfortunately, all cases also contained a varying number of starter neu-

rons in the habenula which impose a limit on the analysis of the affer-

ents to the lateral thalamic group. Some striking differences between

the cases targeting the lateral posterior thalamic Gpr151-Cre popula-

tion and the habenular Gpr151-Cre population could nonetheless be

observed. The cingulate cortical area 1 and area 2, as well as the pre-

limbic cortex, contained many eGFP1 neurons in animals with starter

neurons in the lateral posterior thalamic nucleus, but were completely

devoid of eGFP1 cells when the injections were restricted to the habe-

nula. Similarly, other cortical areas such as the retrosplenial dysgranular

cortex as well as the primary and secondary visual cortex and primary

and secondary somatosensory cortex, only contained eGFP1 neurons

in cases with lateral posterior thalamic starter neurons.

FIGURE 5 Omission of RG limits eGFP expression to the injected area. Coronal brain sections from 1.34 to24.84 mm relative to Bregma of aGpr151-Cre mouse (case M118) in which AAV8-Ef1a-FLEX-TVA-mCherry and SADDG-eGFP(EnvA) was injected unilaterally in the habenula. SinceAAV8-CA-FLEX-RG was omitted, transsynaptic transport of the rabies vector is inhibited. Although many eGFP positive (green) neurons could beseen in various nuclei in the targeted area (medial and lateral habenula, lateral posterior thalamic nucleus, parafascicular thalamic nucleus, paraventric-ular thalamic nucleus, and precommissural nucleus), no eGFP expressing neurons were detected elsewhere in the brain. Scale bar51000 mm


| 3241

Similar to what was observed when the habenula was targeted,

many eGFP1 neurons could be observed in the bed nucleus of the

anterior commissure, nucleus of the diagonal band, ventral pallidum,

bed nucleus of stria terminalis, lateral preoptic area, lateral hypothala-

mic area, and the entopeduncular nucleus. The caudate putamen also

contained eGFP1 positive neurons in contrast to habenula-targeting

injections. Large number of eGFP1 neurons was observed in the zona

incerta, mainly in the caudal part.

In the thalamus, dense populations of eGFP1 neurons were

observed in pregeniculate, dorsolateral geniculate, and medial genicu-

late nuclei as well as the reticulate and paraventricular thalamic nuclei.

In the midbrain, great numbers of eGFP1 neurons were detected in

the superior colliculus as well as in the pretectal region. In the hind-

brain, only a few eGFP1 neurons were detected in the laterodorsal

tegmental nucleus.

4 | DISCUSSION

In this study, monosynaptic tracing using pseudotyped rabies virus was

used to identify primary afferents to populations of Gpr151-Cre neu-

rons in the mouse diencephalon.

In the medial habenula, TVA-mCherry1/eGFP1 starter neurons

were observed in the ventral part of the medial habenula (Table 2).

This distribution is consistent with the pattern of Gpr151 mRNA and

protein expression (Figures 2 and 3). Several nuclei in the pallidum,

including the triangular septal nucleus, septofimbrial nucleus, bed

nucleus of the anterior commissure, medial septal nucleus, and the

nucleus of the diagonal band have previously been identified as affer-

ents to the medial habenula (Herkenham & Nauta, 1977; Qin & Luo,

2009; Yamaguchi, Danjo, Pastan, Hikida, & Nakanishi, 2013). All of

these areas contained eGFP1 neurons in cases where starter neurons

were found in the medial habenula, although the most prominent and

consistent projection appeared to arise in the nucleus of the diagonal

band and the medial septal nucleus. This projection is reported to be

GABAergic (Contestabile & Fonnum, 1983; Qin & Luo, 2009), although

we did not test whether the neurons that project onto Gpr151-

expressing cells have this phenotype.

In the lateral habenula, Cre expression could be detected in all sub-

nuclei except for the oval subnucleus of the lateral division (Figures 2

and 3; Table 2). Afferent neurons were observed in many areas (Figure

7; Table 3) that have previously been reported to contain afferents to

the lateral habenula, including the nucleus of the diagonal band, bed

FIGURE 6 Afferents of habenular Gpr151-Cre neurons. Coronal brain sections from 1.98 to 24.96 mm relative to Bregma of a Gpr151-Cre mouse (case M80) where AAV8-Ef1a-FLEX-TVA-mCherry, AAV8-CA-FLEX-RG and SADDG-eGFP(EnvA) was injected unilaterally in thehabenula. Neurons coexpressing eGFP and mCherry (starter neurons; magenta outline) were almost exlusively located in the medial and lat-eral habenula. EGFP positive neurons (green) were found throughout the brain, most notably in the medial septal nucleus, nucleus of thediagonal band, lateral preoptic area, and the lateral hypothalamus (peduncular part). Scale bar51000 mm


BROMS ET AL.

nucleus of stria terminalis, ventral pallidum, lateral preoptic area, lateral

hypothalamic area and entopeduncular nucleus and the median raphe

nucleus (Dong & Swanson, 2004; Herkenham & Nauta, 1977; Wallace

et al., 2017).

In a recent study, the nucleus of the diagonal band was shown to

provide a possible source of input to the lateral habenula, which

allowed the habenular neurons to phase lock with hippocampal theta

oscillations during anesthesia and rapid-eye-movement (REM) sleep

(Aizawa et al., 2013). Lesions of the lateral habenula and the fasciculus

retroflexus reduce theta power in the hippocampus and substantially

decrease REM sleep duration, while non-REM sleep remains unaf-

fected (Aizawa et al., 2013; Valjakka et al., 1998). Since Gpr151-Cre

neurons receive input from the nucleus of the diagonal band and in

turn project to the interpeduncular nucleus and median raphe, struc-

tures that has been implicated in theta oscillations and REM sleep

(Funato et al., 2010; Vertes, Kinney, Kocsis, & Fortin, 1994), it is possi-

ble that this neuronal population (or a subset of it) plays a role in con-

trolling these functions.

A fruitful approach for determining the valence of experiencing acti-

vation or inhibition of specific neurons is by optogenetic stimulation,

FIGURE 7 Main afferent neuronal populations of the habenular Gpr151-Cre starter neurons. Coronal sections showing eGFP-expressingafferent neurons (green cell bodies and fibers) in the medial septal nucleus and the nucleus of the diagonal band (a; case M17), the lateraland medial preoptic area (b; case M18), the bed nucleus of stria terminalis and the paraventricular thalamic nucleus (c; case M17), the trian-gular nucleus of the septum and the bed nucleus of the anterior commissure (d; case M17), the entopeduncular nucleus and the lateralhypothalamus (e, f; case M18). Scale bar5100 mm


| 3243

combined with the real-time place preference test. In this test, the animals

may freely choose to enter or avoid an area where the laser light is acti-

vated. It has been shown that activation of projections from the lateral

hypothalamic area and entopeduncular nucleus to the lateral habenula is

aversive, that is, the animals avoid stimulation (Shabel, Proulx, Trias, Mur-

phy, & Malinow, 2012; Stamatakis et al., 2016). In line with this finding,

inhibition of the projection from the lateral hypothalamic area to the lateral

habenula using an inhibitory light-sensitive chloride channel, or activation

of inhibitory afferents from the ventral tegmental area, was appetitive (Sta-

matakis et al., 2013). Stimulation of lateral habenular efferents to the ros-

tromedial tegmental nucleus also produced an aversive response

(Stamatakis & Stuber, 2012). It was recently demonstrated that activation

of a projection from the paraventricular thalamic nucleus to accumbens

nucleus evoked the similar avoidance behavior (Zhu, Wienecke, Nachtrab,

& Chen, 2016). Given that the aforementioned projections were observed

in our study, it is tempting to speculate that modulation of GPR151-

expressing neurons could alter the affective component of an experience.

Indeed, selective ablation of Gpr151-Cre neurons resulted in increased

anxiety in the elevated plus maze and open field tests (Kobayashi et al.,

2013).

Two distinct populations of entopeduncular neurons project to the

lateral habenula (Wallace et al., 2017). Glutamatergic parvalbumin-

expressing neurons preferentially targets the oval subnucleus of the

lateral division of the lateral habenula, while a GABA/glutamate core-

leasing somatostatin-expressing population display a more distributed

terminal pattern within the lateral habenula. Given that no Gpr151-Cre

was observed in the oval subnucleus, the entopeduncular afferents

may belong to this population of somatostatin-expressing neurons.

4.1 | Similarities and differences between habenularand thalamic Gpr151-Cre populations

It is clear from the efferent connectivity pattern that Gpr151-Cre neu-

rons of the habenula and the paraventricular thalamic nucleus are dis-

tinct populations. The Gpr151-Cre neurons in paraventricular nucleus

FIGURE 8 Afferents of Gpr151-Cre neurons in the paraventricular thalamic nucleus. Coronal brain sections from 1.98 to 24.84 mm relative toBregma of a Gpr151-Cre mouse (case M113) where AAV8-Ef1a-FLEX-TVA-mCherry, AAV8-CA-FLEX-RG, and SADDG-eGFP(EnvA) was injectedinto the anterior part of the paraventricular thalamic nucleus. Afferent eGFP expressing neurons (green) were found in great numbers in the zonaincerta, medial preoptic area, medial and lateral hypothalamus and to a minor extent in the prelimbic cortex, accumbens nucleus, lateral preopticarea, bed nucleus of stria terminalis, and periaqueductal gray. Neurons coexpressing eGFP and mCherry (starter neurons; magenta outline) werelocated in the paraventricular thalamic nucleus. Scale bar51000 mm


BROMS ET AL.

FIGURE 9 Main afferent neuronal populations of Gpr151-Cre starter neurons in the paraventricular thalamic nucleus. Coronal sections (caseM113) showing eGFP-expressing afferent neurons (green cell bodies and fibers) in the prelimbic cortex (a, left hemisphere), the septohypothala-mic nucleus (b), the median preoptic nucleus (b), the periventricular hypothalamic nucleus (b, d), the medial preoptic area (b), the zona incerta (c,right hemisphere), the anterior hypothalamic area (d), the retrochiasmatic area (d), the peduncular part of lateral hypothalamus (e, right hemi-sphere), the ventromedial hypothalamic nucleus (e, right hemisphere), and the arcuate hypothalamic nucleus (f). Scale bar5100 mm

FIGURE 10 Efferent projections of Gpr151-Cre neurons in the paraventricular thalamic nucleus. Coronal sections of a Gpr151-Cre mouseinjected with AAV8-hSyn1-FLEX-mCherry in the paraventricular nucleus of the thalamus (case M74) showing 3,3’-diaminobenzidineenhanced mCherry immunostaining in cell bodies in the injected area and in efferent projections to the zona incerta (a), the prelimbic area(b), the shell and core of accumbens nucleus (c), and the basolateral amygdala (d). Scale bar51000 mm


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project to the accumbens nucleus, zona incerta, basolateral amygdala,

and prelimbic area, a pattern that is distinct from the mesencephalic

projection of the habenular GPR151-expressing neurons (Figure 10;

Broms et al., 2015). On the other hand, many similarities in afferent

connectivity could be noted between the habenular and paraventricular

thalamic Gpr151-Cre populations. Both populations were targeted by neu-

rons in the bed nucleus of stria terminalis, ventral pallidum, nucleus of the

diagonal band, lateral preoptic area, and lateral hypothalamic area. How-

ever, some striking differences in afferent connectivity were observed. The

zona incerta produced a heavy projection onto paraventricular thalamic

Gpr151-Cre neurons, while habenular afferents from this area were sparse.

The same held true for the medial preoptic area. In contrast to the habenu-

lar population, afferents to the paraventricular thalamic population could

not be detected in the bed nucleus of the anterior commissure, entopedun-

cular nucleus, or in the triangular septum.

The paraventricular nucleus has been implicated in food and drug

reward seeking (reviewed in Matzeu, Zamora-Martinez, & Martin-

Fardon, 2014). In a recent study, it was demonstrated that inhibition of

a projection from the paraventricular nucleus to the accumbens nucleus

ameliorates symptoms of opiate withdrawal (Zhu et al., 2016). The lat-

eral habenula has been implicated in cocaine-induced avoidance behav-

ior (Jhou et al., 2013) and the medial habenula contributes to nicotine

aversion (Fowler, Lu, Johnson, Marks, & Kenny, 2011). It is therefore an

interesting possibility that, despite differences in connectivity patterns,

the diencephalic GPR151-expressing neurons could be functionally

related by involvement in the modulation of drug seeking behavior.

FIGURE 11 Afferents of Gpr151-Cre neurons in lateral thalamic nuclei. Coronal brain sections from 1.98 to 25.02 mm relative to Bregmaof a Gpr151-Cre mouse (case M119) where AAV8-Ef1a-FLEX-TVA-mCherry, AAV8-CA-FLEX-RG, and SADDG-eGFP(EnvA) was injectedinto the lateral posterior thalamic nucleus. Starter neurons expressing both mCherry and eGFP (outlined in magenta) were not restricted tothe target area, but were also found in the laterodorsal thalamic nucleus, central lateral thalamic nucleus, parafascicular thalamic nucleus,posterior thalamic group, lateral habenula, precommissural nucleus, and medial pretectal nucleus. Afferent eGFP expressing neurons (green)were detected in many areas throughout the brain including the cingulate and prelimbic cortices, bed nucleus of stria terminalis, ventral pal-lidum, reticular thalamic nucleus, lateral preoptic area, lateral hypothalamic area, zona incerta, and superior colliculus. Scale bar51000 mm


BROMS ET AL.

4.2 | Afferent connectivity of the lateral thalamic

Gpr151-Cre population, involvement in visual

processing?

Many neurons in the lateral posterior nucleus, lateral dorsal nucleus,

and posterior group of the thalamus displayed Cre recombinase activity

(Table 2; Figure 4m–o). In animals where the viral vector injections

were targeted on the lateral posterior nucleus, there was always some

degree of spread in to the habenula. This precludes clear conclusions

to be drawn from these experiments. However, some characteristics of

the eGFP1 neuronal distribution nonetheless provide clues regarding

the connectivity and function of this thalamic population. Compared to

the afferents of the habenular Gpr151-Cre neurons, a large number of

afferents to lateral thalamic Gpr151-Cre neurons were detected in pre-

frontal cortical areas (primarily the cingulate cortex), the visual cortex,

the caudate putamen, the reticular nucleus, and most notably in the

zona incerta and the superior colliculus (Figure 11; Table 3).

Several studies have previously implicated the lateral posterior tha-

lamic nucleus, the superior colliculus, and the zona incerta in visual

information processing (Benevento & Fallon, 1975; Gale & Murphy,

2014; Krauzlis, Lovejoy, & Z�enon, 2013; Legg, 1979; Power, Leamey, &

Mitrofanis, 2001). Interestingly, it has been demonstrated that the lat-

eral posterior thalamic nucleus contains multimodal neurons that are

sensitive to both pain and light (Noseda et al., 2010). To clarify if

GPR151 is involved in the processing of visual or somatosensory infor-

mation will require further investigation.

4.3 | Cre-independent expression of viral constructs

In wild type mice injected with Cre-dependent viral vectors, we occa-

sionally saw TVA-mCherry and eGFP expression in the vicinity of the

injection tract. This finding suggests that there is a certain degree of

leakiness in the FLEX switch, but that this ectopic expression of TVA-

mCherry (and presumably also RG) is not strong enough to enable

transsynaptic transport of rabies virus particles. This problem has also

been noted by other researchers using the same pseudotyped rabies

tracing system (Callaway & Luo, 2015; Pollak Dorocic et al., 2014). In

Cre-expressing animals, neurons that are TVA-mCherry-positive/eGFP-

positive as a result of leakiness are impossible to distinguish from true

starter neurons. We can therefore not conclude that all TVA-mCherry-

positive/eGFP-positive neurons in the vicinity of the injection tract are

actual starter neurons. Nevertheless, since no neurons were observed

outside of the injected area in wild type mice, the Cre-independent

expression does not affect the validity of the analysis of long-range

projections.

4.4 | Different sensitivity of the methods used for

Gpr151 detection

There are notable differences in expression pattern of GPR151 protein,

Gpr151 mRNA, and Gpr151 promoter activity. Immunohistochemistry

reveals strong GPR151 protein expression in habenular projections

(Broms et al., 2015). In situ hybridization detects Gpr151 mRNA in the

habenula, but also in certain thalamic areas (Figure 3; Berthold et al.,

2003; Ignatov et al., 2004; Lein et al., 2006; Vassilatis et al., 2003). The

expression of Cre-dependent reporters found in the current study

largely corresponds to the mRNA distribution. One exception is the lat-

eral posterior thalamic nuclei, in which only very few neurons express-

ing Gpr151 mRNA has been detected using in situ hybridization (Figure

3c,d; Lein et al., 2006), but ample Cre-dependent mCherry expression

was seen in the current study (Figures 2, 3, and 4m–o). A possible

explanation for this discrepancy is that there is a difference in the sen-

sitivity of the different detection methods. The constructs for the viral

reporter employ strong promoters and enhancer sequences (wood-

chuck hepatitis virus posttranscriptional regulatory element) for

improved expression, elements that the endogenous Gpr151 gene is

lacking. Furthermore, the reporter constructs are permanently acti-

vated by Cre recombination, meaning that even a transient surge in

Cre expression will result in persistent tagging of that neuron. As long

as a no specific GPR151 agonists or antagonists are available, there is

no gold standard for assessing functional GPR151 expression. In the

future when such substances have possibly been discovered, the Cre-

expressing populations described in this study should be examined

electrophysiologically, in order to verify that GPR151 is expressed at

physiologically relevant levels.

4.5 | Caveats of using BAC transgenic Cre mice

Gpr151 mRNA and protein could be detected in many, but not all, neu-

rons that exhibited Cre recombinase activity. One possibility is that Cre

mediated activation of the reporter construct can occur even at low or

transient Gpr151 promoter activation. This would result in persistent

reporter expression, while GPR151 protein and Gpr151 mRNA would

remain undetectable. Although the Gpr151-Cre BAC construct is large

(108 kbp) and should contain most of the regulatory elements of the

endogenous Gpr151 gene, we cannot rule out that the BAC transgenic

approach used in this study suffers from ectopic Cre expression inde-

pendent of the Gpr151 promoter. This could be the result of inserting

the BAC transgene into the vicinity of a strong promoter or enhancer

(Liu, 2013). Ectopic expression could also arise if the endogenous

Gpr151 promoter is epigenetically silenced, while the Gpr151 pro-

moter of the BAC construct remains active. In future studies, knock-in

animals targeting the endogenous Gpr151 gene could be used to mini-

mize the risk of ectopic Cre expression.

5 | CONCLUSION

Here, we present a map of the afferent connectivity of a diencephalic

neuronal population defined by Gpr151 expression. The connectivity

of thalamic versus habenular Gpr151-expressing neurons differed sub-

stantially, indicating a neuroanatomical heterogeneity within the

Gpr151-system. In depth knowledge of the connectivity of this, neuro-

nal population will facilitate the design of investigations regarding the

function of GPR151 as well as the interpretation of the effects of phar-

macological modulators of this system.


| 3247

ACKNOWLEDGMENTS

We sincerely thank Maria Ekemohn, Alice Lagebrant, Joakim

Ekstrand, and Martin Lundblad for valuable input on the manuscript.

This work was supported by the Swedish Research Council, the

Royal Physiographic Society of Lund, Stiftelsen Professor Bror Gade-

lius minnesfond, Gyllenstiernska Krapperupsstiftelsen, and Stiftelsen

Ellen och Henrik Sj€obrings minnesfond.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTION

All authors had full access to all the data in the study and take

responsibility for the integrity and presentation of the data. Study

concept and design: JB, MG, TB, and AT. Acquisition of data: JB,

MG, and LH. Analysis and interpretation of data: JB, LH, and AT.

Writing of the manuscript: JB and AT. Conceptualization and critical

revision of the manuscript for intellectual content: AT and KM.

Obtained funding: JB and AT. Study supervision: AT.

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How to cite this article: Broms J, Grahm M, Haugegaard L,

Blom T, Meletis K, Tingstr€om A. Monosynaptic retrograde trac-

ing of neurons expressing the G-protein coupled receptor

Gpr151 in the mouse brain. J Comp Neurol. 2017;525:3227–

3250. https://doi.org/10.1002/cne.24273


BROMS ET AL.

https://doi.org/10.1002/cne.24273

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